Abnormal left-right organizer and laterality defects in Xenopus embryos after formin inhibitor SMIFH2 treatment

Left-right symmetry breaking in most studied vertebrates makes use of so-called leftward flow, a mechanism which was studied in detail especially in mouse and Xenopus laevis embryos and is based on rotation of monocilia on specialized epithelial surface designated as left-right organizer or laterality coordinator. However, it has been argued that prior to emergence of leftward flow an additional mechanism operates during early cleavage stages in Xenopus embryo which is based on cytoskeletal processes. Evidence in favour of this early mechanism was supported by left-right abnormalities after chemical inhibition of cytoskeletal protein formin. Here we analyzed temporal dimension of this effect in detail and found that reported abnormalities arise only after treatment at gastrula-neurula stages, i.e. just prior to and during the operation of left-right organizer. Moreover, molecular and morphological analysis of the left-right organizer reveals its abnormal development. Our results strongly indicate that left-right abnormalities reported after formin inhibition cannot serve as support of models based on early symmetry breaking event in Xenopus embryo.


Introduction
Left-right symmetry breaking requires in most model vertebrates cilia-driven leftward fluid flow during neurulation and early somitogenesis stages [1]. Leftward flow is produced by rotating monocilia and has been shown to be located on the ventral surface of the gastrocoel roof plate (GRP) of amphibian, mammalian posterior notochord or related structure in zebrafish [2][3][4][5], areas designated as left-right organizer (LRO) [1]. Further studies indicate involvement of leftward flow in LR symmetry breaking of sea urchin [6]. In the next step the flow is transformed into asymmetric molecular processes mediated by immotile cilia located on lateral edges of LRO also known as somitic GRP in frogs [7] and crown cells in the mouse [8]. The immotile cilia are involved in sensing of leftward flow by PKD2 channel mediated calcium influx which in turn regulates at different levels nodal inhibitor dand5 (coco) [7,9]. However absence of structurally equivalent ciliated left-right organizer in chick [10][11][12] and pig [12,13]  as well as in reptiles [14] does not support the universal role of the ciliary flow in vertebrates left-right symmetry breaking. Hence, alternative mechanisms at stages prior to the leftward flow also referred to as "early" were discussed as evolutionary conservative mechanisms [15][16][17]. Particularly serotonin and H + /K + -ATPase which are required for correct LR symmetry breaking in Xenopus have been proposed to break left-right symmetry by generating asymmetric ion flux at early stages [15,18]. However, subsequent studies revealed that both H + /K + -ATPase subunit ATP4a and serotonin are required for correct ciliation suggesting involvement as upstream component in leftward flow [19,20], although the role of serotonin is still a matter of contradiction [21,22]. Alternatively, cytoskeleton based chiral processes were proposed to be basal and to operate already at the single cell level [23,24]. The involvement of cytoskeleton has been supported by studies which have shown that chiral flow of myosin cytoskeleton is required in LR symmetry breaking in C. elegans [25] and that actin associated protein formin is critically involved in correct left-right patterning in snails [26,27]. Moreover, formin perturbation achieved by small molecular compound SMIFH2 causes abnormalities of morphological left-right asymmetry also in Xenopus laevis hence indicating that conservative chiralitybased mechanism of left-right symmetry breaking may operate at cleavage stages parallel to the flow based mechanism [27]. To clarify this issue, we investigated here the effect of formin inhibition in more detail, i.e. using the same inhibitor in different temporal contexts and analyzed how inhibition affects molecular left-right patterning, morphological laterality as well as the left-right organizer. Our data indicate that reported effect of chemical inhibition by SMIFH2 is caused by disturbed morphogenesis of the gastrocoel roof plate and disrupts thereby an upstream component of the flow based mechanism.

Embryo culture and embryo explants
Xenopus laevis embryos were obtained by hormone-induced egg laying and in vitro fertilization using standard methods [28], de-jellied in 2% L-cysteine solution, pH 8, and then cultured in 0.1X MMR at 14 to 18˚C. The embryos were staged according to the tables of normal development [29]. Before fixation the embryos were liberated from vitelline membrane with forceps. Dorsal explants were prepared at stage 18 after fixation as described by Shook and coworkers [30]. All protocols were developed in accordance with European convention for the protection of vertebral animals used for experimental and other scientific purposes (Strasbourg,1986) and approved by Ethic Committee for animal research of the Koltzov Institut of Developmental biology (approval number 53). Animals were anesthetized with MS222 for in vitro fertilization and tadpoles were anesthetized with MS222 prior to fixation.
Dand5 and Tekt2 PCR products for probe synthesis were amplified from previously published plasmids [9,31].

In situ RNA hybridization
Wild-type or formin-modulated embryos were fixed in 4% formaldehyde in PBS for 2 hours, stored in 100% ethanol at -20˚C. Whole-mount embryos or dorsal explants were processed following modified published protocol [32]. All steps were performed in glass vials. Briefly: fixed samples were rehydrated in 75% ethanol, 50% ethanol and 25% ethanol in PBS with 0.1% Tween-20 (PTw) for 5 minutes each, washed three times in PTw for 5 minutes each, then incubated in 10 μg/ml proteinase K in PTw for 3.5 (dorsal explants) to 17 (tailbud stages) minutes. Digestion was stopped by washing twice in freshly prepared triethanolamine solution (TEA), then in 0.25% and 0.5% acetic anhydride in TEA for 5 minutes each. Samples were washed twice in PTw for 5 minutes each and re-fixed in 4% paraformaldehyde in PTw for 20 minutes at room temperature. Samples were washed four times in PTw for 5 minutes each, in 20% hybridization buffer (HB) in PTw and 100% HB for 10 minutes each and prehybridized in HB at 60˚C for 12 h. Samples were hybridized at 60˚C overnight in probe solution (HB containing 300 ng/ml of antisense RNA probe). Samples were washed in prehybridization solution for 1 hour at 60˚C, then three times in 2x SSC (pH 7) at 60˚C for 20 minutes each, twice in 0.2x SSC (pH 7.0) at 60˚C for 30 minutes each and finally twice in maleic acid buffer (MAB; 0.1M maleic acid, 0.15M NaCl (pH 7.5)) at room temperature for 15 minutes each. Samples were treated with blocking solution (1% blocking reagent Roche/MAB) for 2,5 h at room temperature and then with 0.1% anti-digoxigenin AP antibodies in blocking solution overnight at 4˚C. Samples were washed ten times for 1 h in MAB at room temperature and stained in BM purple substrate (Boehringer) at 4˚C in the dark (a few hours to several days). After staining, samples were fixed in 4% formaldehyde/PBS for 2 hours, bleached in a solution of 0.5x SSC, 5% formamide and 5% H 2 O 2 for 1 to 4 h under bright light to remove pigmentation (for stage 28 embryos), re-fixed in 4% formaldehyde/PBS for 2 hours, transferred to ethanol and then to glycerine for long-term storage at -20˚C. Samples were examined at Olympus SZX9 stereomicroscope (Olympus, Japan).

Immunofluorescent analysis of GRP
Wild-type or formin-modulated embryos at stage 18 were fixed in a solution of 4% formaldehyde in PBS overnight at 4˚C, dorsal explants were prepared. Samples were washed in PBS for 30 minutes, in PTw for 1 h, then in PTw with 20% heat-inactivated goat serum for 2 h, and stained with anti-acetylated tubulin primary mouse antibodies (1:1000 in PTw with 20% serum) overnight at 4˚C. After primary staining samples were washed five times in PBS for 1 h each, in PTw with 20% serum for 2 h, and incubated with secondary anti-mouse antibodies (1:1000 in PTw with 20% serum) overnight at 4˚C. Samples were then washed three times in PBS for 15 minutes each, in PTw for 1 h, and stained with 5ng/ml TRITC-Phalloidin in PTw for 45 minutes at room temperature in the dark, then washes twice in PBS for 1 h each and transferred to 80% glycerine in PBS for long-term storage at -20˚C.
Confocal microscopy of stained samples (focused to the gastrocoel roof plate) was performed on confocal laser-scanning microscope Olympus FluoView FV10i (Olympus, Japan).

Light microscopy
For histological analysis, dorsal explants were fixed in Bouin solution, dehydrated through an ethanol series and 100% acetone, embedded in epoxy resin and sectioned on an ultratome (Tesla, Czech Republic). 2-3 μm sections were stained by 1% toluidine blue and examined with a Axio Imager A1 (Carl Zeiss) microscope.

Scanning electron microscopy
Wild-type or formin-modulated embryos at stage 18 were fixed in 2.5% glutaraldehyde in 0.1M cacodylic buffer overnight, dorsal explants were prepared. Samples were postfixed in OsO 4 , dehydrated through an ethanol series and 100% acetone, critical point dried. Dried specimens were mounted on taps with conducting silver and sputtered with Gold-Palladium. Samples were examined and photographed with a CamScan S-2 microscope.

Morphometric measurements and data analysis
Morphometric measurements were made with the open-souce program ImageJ version 1.37 (http://rsb.info.nih.gov./ij/). Statistical significance was determined using the R program (R Development Core Team, 2004).
We determined cilia parameters of GRP cells as described in [20]. A square at the center of the GRP was selected in SEM pictures for manual analysis of cilia length and polarization (posterior, central, other). The ciliation rate was calculated as the ratio of cilia over cells (separately in each GRP SEM photograph). Quantitative data from ImageJ measurements were analyzed with pairwise t test with Bonferroni correction.
To analyze nodal1 and pitx2 expression in stage 28 embryos and heart and gut asymmetry in tadpoles we divided the observed phenotypes into groups and counted the number of samples in each group. Statistical calculations of gene expression patterns and visceral organs asymmetry of formin-modulated embryos were performed with two-proportions Z-test with Bonferroni correction.

Formin inhibition at gastrula-neurula stages disturbs molecular laterality and organ situs
Considering controversial data concerning lethal dosage [27,33,34] we first tested the optimal concentrations of the formin inhibitor SMIFH2. Embryos were treated either during cleavage or during gastrula-neurula stages and the proportion of embryos survived to the tailbud stage (NF stage 28) was used as a read out. SMIFH2 at a 50 μM concentration reveals a general toxicity regardless of treatment stage and resulted in 100% lethality (S1A and S1B Fig). In order to define the developmental period when formins are required for laterality establishment, we treated embryos at stages 2-6,5 NF (during cleavage) and at stages 10-18 (gastrula-neurula) ( Fig 1A). The embryos were then analyzed for nodal1 and pitx2 expression patterns at stage 28 ( Fig 1B and 1C). We subdivided detected nodal1 expression patterns into 4 types: the expression at the left side which correlates with normal laterality, as well as right-sided, bilateral or absent expression (Fig 1B').
The embryos treated with SMIFH2 during cleavage showed no significant change of nodal1 expression, whereas SMIFH2 administration at gastrula-neurula stages led to a dose-dependent decline of the proportion of embryos with left-asymmetric nodal1 expression pattern which was significant for 10μM concentration of inhibitor (Fig 1B).
Similarly, to nodal1, we observed four expression patterns of pitx2 (left-asymmetric, rightasymmetric, bilaterally symmetric and absent). Again, incubation of Xenopus embryos with formin inhibitor SMIFH2 at cleavage stages did not significantly affect pitx2 expression, and formin inhibition in embryos at gastrula-neurula resulted in significantly lower proportion of left-asymmetric patterned embryos for higher dose of inhibitor (Fig 1C). At stage 28, we did not observe any specific features of phenotype in treated embryos.
Since asymmetric molecular patterning is followed by morphological asymmetry in the course of development, we wondered whether the observed effects of formin inhibition would persist in organ laterality of tadpoles and repeated formin inhibition in embryos during cleavage or gastrula+neurula stages, now allowing them to grow up to stage 46. The phenotypes of tadpoles were analysed for heart and gut laterality and classified into situs solitus, situs inversus and heterotaxy (Fig 2A). The analysis of laterality of tadpoles which underwent incubation in formin inhibitor during cleavage does not reveal differences from the control group, whereas the tadpoles exposed to inhibitor at gastrula-neurula stages showed a significantly lower proportion of embryos with situs solitus after a higher dose of inhibitor (Fig 2B).

Formin inhibition perturbs GRP region
As the effect of formin inhibitor is revealed after treatment at the stages of gastrula and neurula, we focused our attention on the so-called left-right organizer which emerges in the GRP at this time. First we studied whether the modulation of the formin activity influences the molecular anatomy of the left-right organizer and analyzed expression of endodermal marker sox17 [35] and of medial GRP marker tekt2 [31] after SMIFH2 treatment. Analysis of sox17 expression in control embryos reveals negative area of characteristic triangular shape at the posterior dorsal midline of embryo, corresponding to the zone of LRO at this time of development ( Fig 3A, S2C Fig). In inhibitor-treated embryos this negative area displays slit-like shape indicating narrowing of surface of LRO (Fig 3A', S2C Fig). Expression of tekt2 reveals a complementary pattern with triangular LRO-related domain in control embryos and slit-like domain in treated embryos (Fig 3B and 3B', S2D Fig). Taking together treated embryos reveal excessive covering of GRP with sox17-positive cells which is accompanied by narrowing of its medial area expressing marker of cilia tekt2. Complementary pattern of tekt2 and sox17 expression is already seen at late gastrula stages while the expression pattern of both markers in SMIFH2 treated embryos is at this stage similar to the DMSO control (S2A and S2B Fig).
Next we analyzed markers of lateral, somitic GRP. The embryos were treated with 10 μM concentration of formin inhibitor during cleavage or at the stages of gastrula-neurula, as described before, and then analyzed for nodal1 expression at the stage 18. nodal1 expression was seen both in SMIFH2 treated and control embryos while embryos treated at gastrula-neurula stages display weaker expression (Fig 3C and 3C', S3A Fig) In the next step, we examined the morphology of the GRP (Fig 4A-4C) in treated and control embryos at stage 18, when all morphological characteristics of the ciliary organizer reach their maximum. SMIFH2 treated embryos reveal at stage 18 phenotypic peculiarity displaying a brick-like shape while the embryos do not fill the space bounded by vitelline membrane.

PLOS ONE
Left-right organizer and formin inhibition SEM images of GRP in Xenopus embryos demonstrate that in comparison to control group the central area of GRP in treated embryos is notably narrower revealing slit-like shape and its lateral sides are covered by larger cells (Fig 4A).
This arrangement can be visualized by immunofluorescent analysis of GRP which reveal a central area covered by small cells bearing long mostly polarized monocilia (S5 Fig) and transverse histological sections through GRP which suggest that the lateral area of LRO in treated embryos is partially covered by endodermal cells (Fig 4B). Importantly, SMIFH2 treatment does not affect cortical actin (S5 Fig). Since the medial areas of the GRP were only incompletely covered by endoderm we were able to study ciliation features: the percentage of ciliated cells at the dorsal midline (measured in the uncovered area) insignificantly declined in treated embryos, and the polarization of ciliated cells judged on the posterior position of cilia did not change, neither did the cilia lengths (Fig 4C and S6 Fig).
Finally, we analyzed morphology and expression pattern of GRP-related genes in embryos treated at cleavage stages and found no signs of endodermal covering of left-right organizer at stage 18 (S7 Fig).

Left-right perturbation after treatment with formin agonist IMM-01
To address specificity of SMIFH2 effect we treated embryos at cleavage or at gastrula-neurula stages with formin agonist IMM-01 and analyzed nodal1 and pitx2 expression at stage 28 as well as the morphology at stage 18. Whereas administration of 10 μM of IMM-01 at cleavage stage was not followed by significant changes of nodal1 or pitx2 expression, treatment with 100 μM led to significant decrease of (left-sided) asymmetry of nodal1 expression. In embryos treated at gastrula-neurula stages treatment with 100 μM led to significant decrease of both

Discussion
Our study aimed to analyze the temporal dimension and morphological details of previously reported [27] effect of chemical formin inhibition on left-right symmetry breaking and patterning in Xenopus. We found that SMIFH2 administration perturbed morphological laterality of visceral organs, the molecular patterning studied by expression of nodal1 and pitx2 in the lateral plate mesoderm as well as the structure of the left-right organizer. Hence, presented data supports and extends reported effect of SMIFH2 treatment on left-right symmetry breaking in Xenopus. Though, our analysis reveals that the significant effect of the SMIFH2 administration was observed only after treatment at gastrula-neurula stages and is dose-dependent. Furthermore, our data is consistent with early findings [33,34], suggesting that SMIFH2 in concentrations 50μM used in [27] is lethal to embryos regardless to development stage, a difference which may be explained by intracellular SMIFH2 breakdown or inactivation [34].
Results of our investigation suggest that reported abnormal development of situs in Xenopus after SMIFH2 administration cannot be used in favour of proposed LR symmetry breaking during cleavage stages. The molecular mechanism of observed effect remains open since SMIFH2 has been shown additionally to modulate myosin retrograde flow and contractility [36]. The specific role of formin in left-right symmetry breaking is supported by situs abnormalities after gain of function experiment performed by microinjection of mouse Dia1 formin into Xenopus animal pole and in dorsal left or ventral right blastomeres [27]. However, reported experimental setting does not allow to study the temporal dimension of effect and cannot be used to discriminate between early and late hypothesis. Importantly, descendants of ventral blastomeres are involved in morphogenesis of the posterior archenteron roof and somite formation and may therefore disturb the function of the left-right organizer [37][38][39]. Treatment of Xenopus embryos with formin agonist IMM-01 led to significantly abnormal nodal1 pattern in the lateral plate mesoderm irrespective of the stage of treatment and therefore does not allow to discriminate between early versus late effect. However, our data indicates that effect of IMM-01 treatment is not due to endodermal covering of GRP and requires an alternative mechanism interfering with left-right patterning. SMIFH2 and IMM-01 target different parts of formin protein: while SMIFH2 binds to FH2 domain of formin molecule [33], IMM-01 disrupts the binding of DID-DAD domains and prevents the formin autoinhibition [40]. Whether observed shortening of cilia disturbs the leftward flow and thereby provides a possible mechanism of effect of IMM-01 remains to be clarified.
Our analysis of morphology and expression pattern of GRP after SMIFH2 treatment revealed extended lateral to medial covering of the left-right organizer surface by endoderm. The extent of the covering varies and the observed effect can be explained by both disturbed flow and disturbed flow sensing. Most of SMIFH2-treated embryos still keep open medial GRP area which bears correctly polarized monocilia. Hence, taking into account that the ciliary organizer with radically reduced number of cilia is still able to produce effective leftward flow [41] a most parsimonious explanation would be a disabled sensing of the leftward flow and further propagation of left-right signaling. Reduction or loss of somitic GRP has been reported in embryos with inhibited FGF signaling [42]. However, this observation was accompanied by disappearance of nodal1 expression domain while in SMIFH2 treated embryos nodal1 domain was suppressed but still detectable. Attenuation of nodal1 expression compared to unaffected expression of dand5 in SMIFH2 treated embryos may be explained by suppression of nodal1 self-enhancement [43] by endodermal covering. An open GRP combined with a strong expression of tekt2, a marker of cilia formation at late gastrula in both control and treated embryos indicate that the covering of GRP in treated embryos at neurula stage is due to a premature closure of the GRP rather than by initially abnormal morphogenesis of GRP. Further studies should determine detailed mechanism of this abnormal morphogenesis.